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Friday, February 19, 2010

MUSHROOM TECHNOLOGY

PROCESS OPTIMIZATION FOR THE PREPARATION OF MUSHROOM (Agaricus bisporus L.) PRESERVE (murabba) AND STUDY OF CHANGES DURING STORAGE









S. No. Chapters Page
1. Introduction
2. Review of Literature
3. Materials and Methods
4. Results and Discussion
5. Summary and Conclusion
6. Literature Cited
Appendix
Vita


Abstract


LIST OF TABLES

Table No. Details of Table Page No.

2.1 Chemical composition of fresh button mushroom (Agaricus bisporus L.)
2.2 Protein digestibility of different food products
2.3 Essential amino acid composition and nutritional value of mushroom protein
2.4 Fatty acid composition of mushroom fat
2.5 Mineral content of Agaricus bisporus L.
2.6 Vitamin content of Agaricus bisporus L.
2.7 Medicinal value of mushrooms
3.1 Particulars of mineral estimation by atomic absorption spectrophotometer
4.1 Physical characteristics of fresh mushroom (Agaricus bisporus L.)
4.2 Proximate composition of fresh mushroom and mushroom preserve
4.3 Mineral content of fresh mushroom and mushroom preserve

4.4 Chemical composition of fresh mushroom and mushroom preserve
4.5

4.6

4.7
4.8

4.9
4.10

4.11
4.12

4.13
4.14
4.15


4.16

4.17
Sensory scores of mushroom preserve prepared using varying periods of blanching
Sensory scores of mushroom preserve prepared using different treatments
Sensory scores of mushroom preserve prepared using different methods
Sensory scores of mushroom preserve prepared using varying concentrations of sugar
Sensory Scores of mushroom preserve prepared using varying concentration of citric acid
Effect of storage time (days) on texture of mushroom preserve stored at room temperature
Effect of storage time (days) on Texture of mushroom Preserve stored at 35⁰C
Effect of storage time (days) on Acidity, pH, TSS of mushroom preserve stored at room temperature
Effect of storage time (days) on Acidity, pH, TSS of mushroom preserve stored at 35⁰C
Effect of storage time (days) on microbiological quality of mushroom preserve stored at room temperature
Effect of storage time (days) on microbiological quality of mushroom preserve stored at 35⁰C


Effect of storage time (days) on Sensory scores of mushroom preserve stored at room temperature
Effect of storage time (days) on Sensory scores of mushroom preserve stored at 35⁰C










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LIST OF FIGURES

Figure No. Details of Figure Page No.

3.1

4.1
4.2
4.3
4.4
4.5
Flow sheet for manufacture of mushroom preserve
Appearance of fresh button mushrooms
Appearance of blanched mushrooms
Appearance of optimized product
Appearance of optimized product stored at room temperature and at 35⁰C
Overall acceptability scores of preserves stored at room temperature and at 35⁰C


CHAPTER-1







Chapter 1 INTRODUCTION

Mushrooms, known as ‘Ksumpa’ in Sanskrit and ‘Khumbi’ or ‘Kukurmutta’ in Hindi are simple form of plant life which have been used by mankind as food and drug since time immemorial. Their use, as recorded in the ancient Indian literature, like the Vedas, dates back to 3000 BC (Singh et al., 1995). The Greeks regarded it as the “Food of God” while Chinese found it as “Elixir of life”. They are not only a privileged class delicacy all over the world but are also the nutritive food especially suited to diabetics.
Mushroom with their great variety of species, constitute a cost-effective means of both supplementing the nutrition to human kind and alleviating the suffering caused by certain kinds of illnesses. The mushroom production in the country, which was around 35,000 tonnes in the year 2001 has increased to about 70,000 tonnes per annum in the year 2006 and is increasing every year. The Indian mushroom industry comprises mainly of small seasonal growers and few export oriented commercial farms having highly technical and environmental control facilities (Dhar, 1997). The mushroom production in India is mainly confined to Himachal Pradesh, Kashmir, Uttar Pradesh, Tamil Nadu and some parts of Haryana, Punjab, Orissa, Karnataka and Delhi (Singh et al., 1995).
Over 2,000 species of mushrooms are known out of which 20 species are cultivated commercially, but only 4-5 species are of industrial significance throughout the world (Chang and Miles, 1991). In India, only 3 species, namely, Agaricus bisporus (European or white button), Pleurotus sajor caju (tropical or oyster or dhingri) and Volveriella volvacea (Chinese or paddy straw) are preferred for commercial cultivation. Of the three cultivated species, the white button mushrooms have the highest consumer preference and account for about 90 per cent of total mushroom production.
They are excellent source of proteins, vitamins and minerals (Prakash and Tejaswin, 1991; Ghosh and Singh, 1995). They posses high potassium : sodium ratio, high dietary fiber, low fat and low calories and have been attributed various medicinal properties. Just like other vegetables, mushrooms contain 90 per cent moisture and are low calorie foods supplying 35 Kcal per 100 g on fresh weight basis. They are low in carbohydrate content, free from cholesterol and have very low amount of fat (0.2 g/100 g). They are also a fairly good source of Vitamin C and Vitamin B complex. They also possess various
therapeutic properties. Further, they offer considerable economic advantage to the grower.
Acceptability of mushrooms in India is limited to small group of consumers because of characteristic flavour, meaty texture and a general suspicious of some of them being poisonous due to lack of awareness. The above situation is further compounded by the fact that 75 per cent of mushroom production comes from marginal or small farmers, who, because of limited resources, grow the Agaricus mushroom during winter months only (Saxena and Rai, 1988). Therefore, it needs proper preservation techniques to promote their consumption among common people and excess mushroom is processed into food products acceptable to consumers.
Mushrooms contain 90 per cent moisture and they do not bear any outer protecting covering therefore rate of respiration from surface is very high (Yappar et al., 1990). These conditions make them highly perishable in nature and hence they can be stored only for few hours under ambient conditions of tropics and subtropics (Rai and Saxena, 1989). They have shelf life of less than a day under ambient conditions (22-28⁰C) (Lal and Sharma, 1995). Surface browning, severe dessication, texture loss are some of the symptoms associated with their
spoilage. Most of the studies on mushrooms, therefore, pertain to increase the shelf life of mushrooms in fresh or processed form and maintenance of their whiteness after harvesting.
Due to its high nutritive and medicinal value, button mushroom is getting popularity day by day and the market potential is increasing considerably. However, its short shelf life due to high rate of metabolism tends to lower its market value and off-season usage. Currently, long term preservation of mushroom by canning and pickling is in vogue. Canning and freezing account for preservation of more than 90 per cent of button mushrooms in India. A number of food products like soup, chutney, ketchup and pickle have been prepared from them (Joshi et al., 1991; Ghosh and Singh, 1995). To further increase the consumption of mushroom and give better remuneration to the growers during the on-season, there is an urgent need for development of various new value added products from mushrooms. One such attempt would be prepare mushroom preserve.
Generally, preserve is made from properly matured fruit, by cooking it whole or in the form of large pieces in heavy sugar syrup, till it becomes tender and transparent. The preserves of aonla, bael, apple, pear, mango, cherry, karonda, strawberry,
pineapple, papaya etc. have gained prime place in market but information regarding the preparation of preserve from mushrooms is very scanty. Therefore, present study entitled “Process optimization for the preparation of mushroom (Agaricus bisporus L.) preserve (murabba) and study of changes during storage” has been planned with the following objectives:
To study the physico-chemical characteristics of white button mushroom.
To optimize the process for the preparation of preserve (murabba) from fresh white button mushroom.
To study the physico-chemical and sensory characteristics of optimized product.
To study the effect of storage on physico-chemical, microbiological and sensory properties.

CHAPTER-2








Chapter 2 REVIEW OF LITERATURE


Intermediate moisture foods have been attracting widespread attention in recent times. They are dry enough to be shelf stable without needing refrigeration or thermal processing in hermetically sealed containers. The technique of intermediate moisture food preparation by immersion equilibration procedure has already applied to guava, pineapple, mango, carrot and banana with satisfactory results.
In India preserves (murabbas) have been used widely both as a delicacy as well as adjuncts in indigenous system of medicine. Some of these preserves are widely recognized locally from their therapeutic role but no effort has been made to correlate these claims with important constituents obtained in these preserves.
2.1 Mushroom
Mushroom has been treated as a special kind of food since ancient times. They have been used as food by human beings since time immemorial. Some reports indicated their potential as life saving drug, antibiotic and biologically active substances. The prospects of growing temperate mushroom in India were first discussed by Padwick (1941).
Mushrooms grown in India broadly fall in two categories: (i) Guchhis and (ii) White Button Mushroom. Guchhis, also known as truffles and morela, are rated as the best quality and are highly priced in the world. These are found in the hill regions of Kashmir valley, Himachal Pradesh, Sikkim, Darjeeling, West Bengal, Arunachal Pradesh, Porhi Garhwal and Kumaun. On the other hand, the “white button mushroom” is a cultivated variety and its commercial production in India commenced in 1961 when Indian Council of Agriculture Research in collaboration with department of Horticulture, Himachal Pradesh took up its cultivation in the state of Himachal Pradesh. Later on, its cultivation spread to other states. Since then, mushroom industry has increased in size by way of expansion of mushroom growing activities all over the country. The mushroom industry saw its biggest expansion in the mid nineties with both production and exports going up dramatically.
Mushrooms are large reproductive structures of edible fungi belonging to class ascomycetes or basidiomycetes. They could be either epigeal or hypogeal. Agaricus bisporus L. belongs to class basidiomycetes, subclass Holobasidiomycetidae, order
Agaricales (Miles and Chang, 1997). Agaricus bisporus L. belongs to Agaricaceae family. The vegetative part of mushrooms mainly consists of thread like long, thin mycelia which, under suitable conditions form fruit bodies or sporocarps.
Mushrooms are non-green fungal plants occurring seasonally all over the world in various habitats varying from sandy plains to thick forest or green meadows to roadside pathways (Chang and Miles, 1991). The mushrooms comprise a large heterogeneous group which differs greatly in their shape, size, colour, appearance and edibility. It has already acquired considerable commercial status in USA, France, Switzerland etc. (Pruthi et al., 1984). The food value of mushroom is being increasingly realized and appreciated by food experts because of its low calories and high nutritive value in terms of vitamins, minerals and protein. Mushroom can serve as protein rich food in developing countries, where the population mainly depends on cereal based foods (FAO, 1972).
2.2 Morphological and physical characteristics
2.2.1 Morphological features
Mushrooms are special group of macroscopic fungi. They lack chlorophyll and therefore, need a substrate for nutrition. Mushrooms produce enzymes that degrade complex organic matter and absorb the soluble substances (Chang and Miles, 1989). A typical button mushroom has mainly three parts namely, Cap (pileus), Gills and Stalk or Stipe. It may or may not have a ring called ‘annulus’ below the cap, and a basal cap or volva (Nichols, 1985).
2.2.2 Physical features
According to Lal and Sharma (1995), the typical features of fresh mushroom are veil closed, gills not visible, upper surface of cap strongly convex and stem plump rather than elongated. Beelman (1988) observed that consumers prefer clean, closed (i.e., gills not open), smooth surface with crisp and tender structure. These characters are associated with freshness of mushroom.
2.2.2.1 Colour
Fresh white button mushrooms are white or light buff without any dark mark on either cap or stem (Lal and Sharma, 1995).
2.2.2.2 Size
Average size of mushroom has been reported to be of 25 to 45 mm in diameter, 3 to 10 mm stipe and 11.5 ± 1.8 g in weight (McArdle et al., 1974; Beelman and McArdle, 1975; Burton et al., 1987; Riva et al., 1991; Lopez-Briones et al., 1992). Average size for export and domestic fresh market button mushroom is generally 20 to 40 mm buttons with velum intact (Nichols and Hammond, 1975 and Dang et al., 1978).
2.3 Chemical composition and nutritive value
The composition and nutritional quality of mushrooms vary with strain, substrate, cultivation technology, stage of harvest and post harvest management (Bano et al., 1988 and Rai, 1990). As food, the nutritional value of mushrooms lies between meat and vegetable and is being considered as vegetable meat. They are excellent source of some vitamins, minerals and essential amino acids (Bahl, 1987; Haque and Chakrabarti, 1987; Jain and Singh, 1983; Sethi and Anand, 1982). It is claimed that mushroom is a protective food, containing most of the essential amino acids in protein which are required for the growth of human body. It is non-fattening and forms an important constituent for the balanced diet.
2.3.1 Proximate composition
With respect to proximate composition, there are wide variations in their values as reported by different workers for the same species and for different species of mushrooms. Proximate composition of various mushrooms has been reported by
Table 2.1: Chemical composition of fresh button mushroom [Agaricus bisporus L.]

Moisture
(%) Protein
(%) Fat
(%) Carbohydrate
(%) Ash
(%) Crude fiber (%) Ascorbic acid (mg/100 g) Reference
A B A B A B A B A B A B
92.8 - 42.5 - - - - - 14.5 - 16.2 5.0 - Pruthi et al.,
(1984)
89.5 - 26.34 - 1.8 - 59.9 - 12.0 - 10.4 - 82 Bano and Rajarathnam (1986)
89.5 - 26.3 - 1.8 - 59.9 - 12.0 - 10.4 - - Bano et al., (1981)
88.6-92.5 3.1-3.9 - 0.88-0.92 - 3.94-4.07 - 0.801-1.40 - 0.73-1.0 - 5.69-6.2 - Tomar,
(1998)
90.0-94.2 3.05-3.8 - 0.29-0.37 - 3.87-4.50 - - - 0.83-1.12 - 5.69-8.3 - Sethi et al.,
(1991)
90.7-92.5 3.5-3.9 - 0.28-0.36 - 3.78-4.36 - 0.96-1.9 - 0.34-1.13 - 5.8-8.69 - Choudhary,
(2000)
91.2 3.85 - 0.18 - 3.63 - - - - - - - Tyagi,
(2004)

Fresh weight basis, B- Dry weight basis


Howard et al., 1962; Bano et al., 1981; Lapedes, 1977; Gopalakrishnan and Pruthi, 1977; Crisan and Sands, 1978; Gopalan et al., 1982; Bano and Rajarathnam, 1982; Jain and Singh, 1983; Sethi and Anand (1982, 1984); Rai and Sohi, 1988; Chang and Miles, 1989 and Naikkhurade et al., 1984. Nutritive value of wild edible mushrooms from Kashmir has been estimated by Khurana et al. (1987). Table 2.1 shows the proximate composition of button mushrooms as reported by various workers.
2.3.1.1 Moisture
Pruthi et al. (1984) reported moisture content between 86.5 to 92.0 per cent in fresh mushroom. The average moisture content of mushrooms is about 90 per cent. Deviations from this value have been reported whenever there is a variation in the culturing conditions such as water content of the bed, temperature and relative humidity of the chamber (Rajarathnam and Bano, 1988 and Bano et al., 1981).
Rai (1990) showed that moisture content of mushroom was affected by the stage of growth, environmental conditions around the fruit body sampled, watering regime and post harvest storage before analysis.

Maggioni et al. (1968) observed that initial moisture content decreased in mushroom from first break to fourth break.
2.3.1.2 Protein
Mushroom proteins are comparable to muscle proteins in nutritive value. In the context of world protein shortage, the Food and Agriculture Organization (FAO) of the United Nations has recommended mushroom as supplementary food item to the growing population of the developing countries, which are based mainly on cereal diet (Hayes and Haddad, 1976). Agaricus bisporus L. contains 2.9 to 3.9 per cent protein on fresh weight basis and 20-40 per cent on dry weight basis (Pruthi et al., 1984; Bano and Rajarathnam, 1986; Tomar, 1998; Sethi et al., 1991 and chaudhary, 2000). Tyagi (2004) found that fresh Agaricus contained 3.85 per cent protein. On fresh weight basis, the lowest value of protein content was found to be 1.8 per cent (McCane and Widdowson, 1960) and highest to be 5.9 per cent. Anderson and Fellers (1942), McConell and Esselen (1947), Hayes and Haddad (1976) and Kaul (1983) reported a range of 3.5 to 3.95 per cent protein in mushroom on fresh weight basis. Maw and Flegg (1975) found an average protein content of 3.5 to 4.0 per cent which is in agreement with the value of 3.7 per cent stated in a FAO publication (FAO, 1970). Weaver et al. (1977) found a considerable variation in protein content of different strains which ranged between 1.91 to 4.11 per cent on fresh weight and 19.4 to 38.8 per cent on dry weight basis. A higher value of 46.5 per cent on dry weight basis was also reported by Abou-Heilah et al. (1987). Only 60 to 89 per cent of mushroom protein (N × 6.25), with an average value of 70 per cent, is digestible. Some of the reported values for per cent protein from India on dry weight basis were 42.25 (Pruthi et al., 1984) and 26.3 (N × 4.38) by Bano et al. (1981). Table 2.2 compares protein quality and digestibility of different food products.
2.2: Protein digestibility of the different food products
Food products Total protein percent (d.b) Digestible protein percent Digestibility percent
Meat
Agaricus sp
Spinach
Legumes
Rye bread
Potatoes 83.7
51.9
34.5
26.3
10.7
8.0 82.8
45.9
25.0
23.4
9.0
7.3 98.9
88.5
72.5
89.0
84.1
91.2
Source: Anderson and Fellers (1942)

Amino acids
Nearly one fifth of the total nitrogen is in the form of free amino acids (Sato et al., 1985 and Wang and Li, 1985). Alanine, glutamate and glutamine were found to be the dominant acids in the free amino acid pool (Sato et al., 1985). Mushroom protein is intermediate in quality between vegetable and animal proteins and the supplementary value of mushroom protein in vegetarian diet is of considerable significance. Nutritionally, mushrooms fall between high grade vegetables and low grade meats and provide about 35 cal/100g fresh weight (Rai, 1995). It is because all the essential amino acids are present in mushroom. Bano et al. (1981) determined the quality of mushroom proteins in Agaricus bisporus L. They reported a value of 43.1 for chemical score (egg as reference protein), 36.1 for chemical score (FAO reference protein); 55.8 for essential amino acid index, and 49.1 for biological value. According to them sulphur containing amino acids are the first limiting amino acids. Weaver et al. (1977) reported 10 g lysine per 100 g of protein in Agaricus bisporus L. on dry weight basis. Essential amino acid composition and nutritional value of mushroom protein has been shown in Table 2.3.

Table 2.3: Essential amino acid composition and nutritional value of mushroom protein

Amino acid/Protein quality units Agaricus bisporus L. Pleurotus florida
(mg/100g)

Leucine 7.5 7.5
Isoleucine 4.5 5.2
Valine 2.5 6.9
Tryptophan 2.0 1.1
Lysine 9.1 9.9
Threonine 6.1 6.4
Phenylalanine 4.2 3.5
Tyrosine 3.8 2.7
Cystine 1.0 0.2
Methionine 0.9 3.0
Arginine 12.1 3.2
Histidine 2.7 2.8
Total essential amino acids 41.6 46.4
Chemical score(egg as reference protein) 43.1 67.4
Chemical score(FAO reference protein) 36.1 71.8
Essential amino acid index 55.8 84.5
Nutritional index 17.6 15.9
Biological value 49.9 80.4




2.3.1.4 Fat
Mushroom is a low fat food (Bano and Rajarathnam, 1986). The fat content of Agaricus bisporus L. ranges from 0.1 to 0.5 per cent on fresh weight basis (Crisan and Sands, 1978). Low fat content, high fiber content and absence of cholesterol makes mushrooms dietician’s choice for heart patients. Agaricus contains 0.18 to 0.39 per cent fat on fresh weight basis (Khaddar et al., 1999). Rai (1990) reported lower range of fat content (0.10 to 0.19 per cent on fresh weight basis) in Pleurotus species. Bano and Rajarathnam (1986) reported 1.8 per cent fat in Agaricus bisporus L. and 1.0 to 9.4 per cent in Pleurotus species on dry weight basis. Crisan and Sands (1978) reported 2 to 8 per cent crude fat in mushroom on dry weight basis. The crude fat of mushrooms include, free fatty acids, mono-, di- and triglycerides, sterols, sterol esters and phospholipids. Tomar (1998); Choudhary (2000) observed higher (0.28 to 0.88 per cent on fresh weight basis) values of fat in Agaricus bisporus L. According to Tyagi (2004), fresh Agaricus contained 0.18 per cent fat. Maggioni et al. (1968) studied fat fraction of Agaricus bisporus L. mushroom and found that most common fatty acids are palmitic acid, stearic acid, oleic acid and linoleic acid. Linoleic acid, nutritionally the most desirable poly unsaturated fatty acid, constituted 70 per cent of fatty acids of neutral lipid fraction and 90 percent of polar lipid fraction. Huang et al. (1985) found that linoleic acid accounted for 76, 70, 69 and 63 per cent of total fatty acids in Lentinus edodes, Volvariella volvacea, Agaricus bisporus L. and Pleurotus Sajor Caju, respectively. Li and Chang (1982) observed that crude fat increases as the paddy straw mushroom matures and constitutes about 5 per cent of the dry weight of fully matured fruit bodies. It appears that the variation in fat content is because of the difference in species, growing conditions and the maturity at the harvesting time.
Huang et al. (1985) studied the fatty acids and sterols of commercial mushrooms. Their most interesting and useful finding was fatty acid distribution in mushroom lipids. At least 72 per cent of total fatty acids were found to be unsaturated. Among the sterols, ergosterol is abundant. Most common sterols found in commercial mushrooms were provitamin D2 (ergosterol), provitamin D4 (22-dihydro-ergosterol) and γ-ergosterol.
Naikkhurde et al. (1984) reported that although fat content in mushrooms is as low as 0.3 per cent but it is rich in linoleic acid, which is an essential fatty acid (Table 2.4). In

Table 2.4: Fatty acid composition of mushroom fat

Fatty acids Agaricus bisporus L.

C10:0 -
C12:0 Traces
C14:0 Traces
C16:0 15.40
C18:0 2.50
C18:1 3.30
C18:2 78.60
Saturated 15.40
Unsaturated 84.40
Polar 50.30
Nonpolar 49.40
Holtz and Schister (1971)
(Values as per cent of total crude fat)



mushrooms cholesterol is replaced by ergosterol which gets converted to vitamin D in human body.
2.3.1.5 Carbohydrates
Bano and Rajarathnam (1982) reported that Pleurotus florida contained 58 per cent carbohydrates on dry weight basis. Carbohydrates content of Agaricus bisporus L. range from 5.0 to 6.5 per cent on fresh weight basis and 50 per cent of these carbohydrates were nitrogen free. Thayumanavan and Manickam (1980) noted 20 per cent carbohydrates in mushroom on dry weight basis. Various workers (Anderson and Feller, 1942; Mc Conell and Essellen, 1947; Tomar, 1998; Chaudhary, 2000) have reported average carbohydrate content of fresh Agaricus bisporus L. between 2.9 and 5.9 per cent. According to Tyagi (2004) fresh Pleurotus contained 4.72 per cent total carbohydrates and fresh Agaricus contained 3.63 per cent. McConnell and Esselen (1947) found that carbohydrates of mushroom are polymeric carbohydrates including glycogen, which serve as a source of energy during storage and is comparable to starch in higher plants. Chitin, a polymer of N-acetyl glucosamine, is the major constituents of its fiber (Rai, 1995).


Hammond (1986) reported that carbohydrate fraction of Agaricus bisporus L. had a large number of compounds like pentoses, methyl pentoses, hexoses, oligosaccharides, amino sugars and sugar alcohols. Among sugars, trehalose ‘the mushroom sugar’, and the sugar alcohol mannitol have been found to play a key role in fruit body formation and its development. Mannitol was found positively associated to yield of flush (Parrish et al., 1976). According to Ajlouni et al. (1992) higher amount of carbohydrates (28 per cent on dry weight basis) are present in lower stipes; gills contained 10 per cent carbohydrates and upper stipes contained 19 per cent carbohydrates. Since mannitol is the main respiratory substrate, they found removal of stipe to result in increased shelf life of mushrooms.
2.3.1.6 Fiber
Fiber content of mushrooms has been reported to be low (0.9 to 1.17 per cent on fresh weight basis). These values did not change much till maturity. However, its level showed a sharp increase on attaining maturity. Some researchers (Tomar, 1998 and chaudhary, 2000) found slightly lower range of crude fiber (0.34 to 1.126 per cent). Bano and Rajarathnam (1986) reported 10.4 per cent fiber on dry weight basis in Agaricus bisporus L. Thayumanavan and Manickam (1980) reported 11.25 per cent fiber content while Pruthi et al. (1984) reported 16.2 per cent fiber content on moisture free basis. The major fraction of fiber in mushroom is ‘chitin’ which is a polymer of N-acetyl glucosamine and is a structural component of cell wall (Rai, 1995).
2.3.1.7 Minerals
Mushrooms supply a number of valuable minerals especially potassium and iron. Minerals like calcium, phosphorus, sodium and copper are also present. Mineral content varies from species to species depending upon cultivated substrate. Mushrooms have higher potassium to sodium ratio. Various workers (Bano et al., 1981; Sethi and Anand, 1984; Rai and Sohi, 1988) observed that the mineral content in fresh mushrooms was higher (0.52-1.7 per cent) than in many fresh fruits (0.2-0.8 per cent) and vegetables (0.6-1.1 per cent) except leafy vegetables.
Chang and Miles (1989) reported 8 to 10 per cent ash on dry weight basis in Agaricus bisporus L. Potassium constituted 45 per cent of total ash content followed by phosphorus, sodium, magnesium and calcium which together constituted about 56 to 70 per cent of total ash content. According to Anderson and
Table 2.5: Mineral content of Agaricus bisporus L.


Mineral Agaricus bisporus L. (mg/100g Fresh weight)
Calcium 0.026-9.0
Phosphorus 90-148
Potassium 320-570
Iron 0.5-1.87
Magnesium 1.32-13.0
Sodium 9-110
Zinc 0.1-7.64
Copper 0.16-0.64




Fellers (1942) calcium and iron are present in low concentrations; one third of total iron is in available form. Table 2.5 represents the mineral content of Agaricus bisporus L. on fresh weight basis.
2.3.1.8 Vitamins
Mushrooms are good source of water soluble vitamins, i.e. vitamins of B and C group, but they are relatively poor in fat soluble vitamins. They are relatively good source of vitamin B complex particularly thiamine, riboflavin, niacin, biotin and pantothenic acid. According to an FAO (2006) report mushrooms contain 2.5 mg/100 g ascorbic acid on fresh weight basis whereas others (Tomar, 1998 and chaudhary, 2000) have found a higher value (5.69 to 8.69 mg/100 g). The thiamine content has been reported to vary from species to species and with the substratum used in their cultivation (Crisan and Sand, 1978 and Chang and Miles, 1991).
Thiamine content of mushroom was estimated to be 0.11 to 0.19 mg/100 g on fresh weight basis (Crisan and Sand, 1978; Chang and Miles, 1991 and chaudhary, 2000). According to Crisan and Sand, (1978) and Chang and Miles (1991) pantothenic acid content of mushroom was 2.38 mg/100 g on fresh weight basis. Riboflavin content was found to be 0.16

Table 2.6: Vitamin content of Agaricus bisporus L.


Vitamin Agaricus bisporus L. (mg/100g dry weight)

Thiamine 1.14
Niacin 36.19
Riboflavin 4.94
Pantothenic acid 4.95
Folic acid 22.80
Ascorbic acid 0.93

Bano and Rajarathnam (1986)


to 0.52 mg/100 g in fresh mushrooms (Crisan and Sand, 1978 and Chang and Miles, 1991). Bano and Rajarathnam (1986) reported the vitamin content of Agaricus sp. as summarised in Table 2.6.
It has been observed that after 4 days of storage A. bisporus L. lost 20-25 per cent vitamin C, loss being greater at 15⁰C and at 5⁰C, and the decrease in ascorbic acid was accompanied with increase in dehydro-ascorbic acid (Rai and Saxena, 1989). Canning and drying resulted in 50-60 per cent loss of vitamins (Sethi and Anand, 1984) in Agaricus. The authors also reported wide variation in vitamin C content of different species of mushrooms.
2.4 Enzymes
Mechanical injury to the tissues during handling, slicing and washing results in rapid browning of tissues. Polyphenol oxidase (PPO) is the main enzyme responsible for this change of colour. Mallitte and Dawson (1949) were the first to report several forms of PPO system in mushroom tissues. This enzyme, in the presence of oxygen catalyze the oxidation of phenolic compounds (the oxidation of O-diphenols and the oxygenation of mono phenols) to O-quinones, which in turn rapidly condense to form complex brown pigments the melanins (Nicholas, 1962;
Long and Alben, 1969). Polyphenol oxidases have been distinguished as tyrosinase, catecholase and DOPA- oxidase enzymes on the basis of their activity on specific substrates. The mushroom skin contained the largest amount of tyrosinase followed by flesh, stalk and gill tissues (Burton, 1988 and Moore and Flurkey, 1989). Tyrosinase is major phenol oxidase in Agaricus bisporus L. (Ratcliffe et al., 1994). Ingebrigtsen et al. (1989) observed the extract of Agaricus bisporus L. mushroom had maximum polyphenol oxidase activity with catechol (15 to 29 units/mg) followed by L-DOPA (4 to 16 units/mg) and tyrosine (1-3 units/mg) at different stages of their growth. According to Reed (1966) catecholase is specific for ortho position of diphenols. Espin et al. (1999) studied kinetics of PPO using substrate γ-L-glutaminyl-4-hydroxybenzene (GHB) and it’s O-diphenol, γ-L-glutaminyl-4-hydroxydibenzyne (GHDB). They found that Vmax of enzyme was 2.1±0.1 µM/min and 210±7.3 µM/min, and Km was 0.3±0.003 MM and 7.8±0.41 MM for GHB and GHDB, respectively. To increase the shelf life of fresh mushrooms, it is necessary to (1) prevent transpiration (2) decrease the rate of respiration (3) prevent exposure to atmospheric oxygen, and (4) overcome PPO activity and prevent polymerization of oxidized phenols into melanoid pigments (Rajarathnam and Bano, 1988).
Mushroom enzymes can be inactivated by use of chemicals, lowering pH etc. (Golan-Goldhirsh and Whitaker, 1984). The simplest method of inactivating polyphenol oxidase is to apply heat (Ponting, 1960).
2.5 Medicinal value
In China and Japan mushrooms have traditionally been used for the medicinal values (Miles and Chang, 1997). Cosmetic products and tonic beverages have also been produced in China from Ganoderma mushrooms. Mushrooms have been attributed with various therapeutic properties. They are reported to be helpful in preventing high blood pressure, atherosclerosis, kidney ailments and diabetes etc. Mushrooms, with their good nutritional and high digestibility value are gaining importance as a health food (Rai and Saxena, 1989). Several edible fungi have been reported to exhibit anti-tumor activity, prominent among them are L. edodes, F.velutipes, P.ostrealus and A. Bisporus L. (Misra, 2003). Bahl (1987) and Sethi and Anand (1984) pointed out the medicinal value of edible fungi including mushrooms having antibacterial, anti-fungal, anti-tumour effects, anti-protozoal, hypolipidemic and anti-viral activities. Table 2.7 shows the medicinal value of different mushrooms as observed by several workers.


Table 2.7: Medicinal value of Mushrooms

Irregularity Reference

Blood pressure Kaneda and Tokuda (1966)
Antibiotic Cochran (1978)
Cancer Manning (1985)
Chronic Renel Failure Tam et al. (1986)
Constipation Rai and Sohi (1988)
Hyper acidity Rai and Sohi (1988)
Diabetes Chang and Miles (1989)
Hypertension NCMRT (1990)
Obesity NCMRT (1990)
Anti – tumour Bano et al. (1981) and
Singh et al. (1995)
Nervous System Regulation Jong and Birmingham (1992)
Atherosclerosis Chadha and Sharma (1995)




2.6 Processing of mushrooms
Mushrooms are highly perishable because of their high moisture content and delicate texture and cannot be stored for more than 24 hours at ambient temperature (Lal and Sharma, 1994; Gothandapani et al., 1997). Rate of respiration of fresh mushroom is very high. This results in rapid deterioration of their quality, loss in texture, development of off flavour and discolouration. They are processed within two days of harvest (Bano and Singh, 1972). Various methods like, dehydration, canning, pickling and freezing have been used for extending their shelf life (Bano et al., 1988 and Singh and Bano, 1997).

2.6.1 Blanching
Riaz et al. (1991) found that blanching sliced (5 mm thick) Pleurotus ostreatus in hot water at 80⁰C for 2 min was adequate to inactivate peroxidase enzyme activity. However, prolong blanching caused a substantial loss of nutrients. For inactivation of peroxidase and catalase, hot water blanching for 3 to 5 minutes was found to be better than steam blanching for 4 to 5 minutes for retaining colour (Pruthi et al., 1984). Coskuner and Ozdemil (2000) blanched Agaricus bisporus L. in solution of citric acid (0.5 to 1 g/l) or EDTA (0.5 to 8.0 g/l). They observed that iron and copper content reduced significantly in EDTA blanching while citric acid blanching was suited to control browning and spoilage prior to processing. According to Sethi et al. (1991) blanching was found to be essential.
2.6.2 Canning
Beelman et al. (1973) standardized process to get maximum canned yield of Agaricus bisporus L. They suggested that mushroom soaked for 20 minutes, stored at 20⁰C for 18 hours and again soaked for 2 hours prior to canning give the best results. Mushrooms were blanched in boiling water till temperature of centre reached to 77⁰C, cooled for 2 minutes, filled in can and processed at 121⁰C for 20 minutes.
McArdle et al. (1974) gave various pre-treatments before canning to get higher canned yield and colour value. They found vacuum soaking (submerging in water, subjecting to 42 mm Hg vacuum, 5 minutes holding under vacuum and subsequently for 10 minutes at atmospheric pressure) to give best colour for canned mushrooms.
Dehydro-canning refers to canning of partially dehydrated product. This method of preservation includes dehydration to a point where product quality is not damaged (Lazor, 1978).

Canning of partially dehydrated vegetable like peas was done by Andreotti et al. (1982).
2.6.3 Dehydration
Zhuk and Tsapalova (1973) recommended a temperature of 60⁰C for mushroom drying. But Pruthi et al. (1978) found that paddy straw mushroom dried best at 70⁰C, 65⁰C and 55 to 60⁰C for a period of 2 hours, 2 hours and 4 hours respectively. Dehydration ratio and rehydration ratio of the dried samples varied from 10.0 to 11.1 and 3.2 to 7.5, respectively. However, Singh (1996) recommended a temperature of 60⁰C for drying. He suggested 7 hours drying time to achieve 5 per cent moisture level.
According to Bonazzi (1991) trans 1-octane 3-ol, which is the main flavouring compound of mushroom, changed to benzaldehyde during the drying process.
Jorege and Chanes (1992) found a temperature of 60⁰C adequate for air-drying of mushrooms. Lal and Sharma (1995) recommended a finishing temperature of not more than 65.5⁰C. Pruthi et al. (1978) demonstrated dehydration of paddy straw mushroom in a phased manner at 70⁰C, 65⁰C and 60⁰C. The loss of moisture was significant during first two hours and
dehydration was almost completed within 7 hours. Drying in phased manner was reported to give better results with respect to colour.
Suresh and Samsher (2004) evaluated the quality of poly house dried mushroom slices. Experiments were conducted to investigate the effect of various pre-treatments and poly house drying on drying characteristics of oyster mushrooms. The temperature in poly house drier ranged from 20-50⁰C against ambient temperature of 12-30⁰C during winters. Results revealed that the moisture content and average drying rate decreased with increase in dehydration time. Mushroom samples pre-treated with combination of blanching plus steeping in KMS (0.5 %) and citric acid (0.25 %) solution rated highest sensory score for overall quality stored at room temperature.
Singh et al. (2007) performed tray drying of button mushroom. Slices of 0.5, 0.7 and 0.9 cm thickness of button mushrooms were dehydrated in tray dryer at 40, 45, 50 and 55⁰C and their drying characteristics such as rate of diffusion and rehydration ratio were studied. The qualities of dehydrated slices were evaluated on the basis of colour, veil opening and amino acid content. The samples dehydrated at 50⁰C showed better quality. The diffusivity varies from 1.05 × 10-8 to 7.48 × 10-9 m2/s and increased with temperature. No samples showed veil opening.
Hammami and Rene (1998) optimized the conditions for vacuum freeze drying of sliced Agaricus bisporus L. Ramachandra and Ramanathan (1978) reported that freeze dried products are better in quality, wholesomeness, rehydration and colour than air dried products. However, it is ten times costlier than the latter method. Martinez et al. (2001) found that quality of freeze dried mushrooms (Pleurotus ostreatus) was superior to those obtained by air drying and vacuum drying.
Yang and Le-Maguer (1992) designed a continuously circulating contacting reactor for osmotic dehydration of sliced mushrooms. The mushroom slices were blanched in 15 per cent solution or dehydrated in 60 per cent sucrose solution for 10 minutes before treatment with the salt solution for optimum removal of water was developed by Singh et al. (1995).
2.6.4 Steeping preservation
Steeping mushrooms in an appropriate solution extends their shelf-life. Pruthi et al. (1984) tried various steeping solutions to preserve blanched and unblanched Agaricus bisporus L. They concluded that steeping water blanched mushrooms in 0.5 per cent citric acid and 500 ppm SO2 was best and keep mushrooms for 3 to 4 weeks. Bano and Singh (1972) studied the effect of common salt, pH and preservatives on the shelf life and quality of steeped mushroom. They used steeping solution of 2.5 per cent common salt, 0.2 per cent citric acid, 0.1 per cent ascorbic acid, 0.1 per cent sodium bicarbonate and 0.1 per cent potassium metabisulphite for extending shelf-life of Agaricus bisporus L. to 10 days at room temperature. Singh (1997), during his investigation on long term storage of mushrooms (Agaricus bisporus L.) found that the best steeping solution for blanched mushrooms contained salt, sugar, ascorbic acid, citric acid and KMS in the concentration of 5, 1, 0.5, 0.3 and 0.1 per cent, respectively. The same observations were also confirmed by Neetu (2001).
2.6.5 Freezing of mushrooms
Czapski and Szudygu (2000) reported that treating Agaricus bisporus L. before freezing in 3000-5000 ppm KMS, blanching in boiling water for 20 sec and storage at -20⁰C for 90 days gave frozen products a good colour and appearance. The residual SO2 of this sample was 52 mg/kg on day 1, which decreased to 42 mg/kg on day 14 and remained on this level during rest of storage. Kondratowicz and Dajanowska (2000) used liquid CO2 to freeze (-70⁰C) Pleurotus ostreatus mushrooms and concluded that mushroom could be stored in refrigerated conditions for more than 90 days.
2.6.6 Irradiation
Roy et al. (2000) found that gamma irradiation at the level of 0.5 KG could be extended the shelf life of Pleurotus sajarcaju up to 90 days at 15⁰C compared to 6 days of control.
Benoit et al. (2000) assessed the effect of gamma irradiation (0.5, 1.5 and 2.5 kGy) on biochemical changes in Agaricus bisporus L. He found that 1.5 and 2.5 kGy dose reduced rate of respiration of mushrooms. The irradiation increased phenylalanine ammonialyase activity and total phenol content between day 1 and 4 then decreased to lower values till end of storage (12 days). While polyphenol activity increased until 7, 9 and 12 days for samples treated at 0.5, 1.5 and 2.5 kGy, respectively.
Singh et al. (1995) employed gamma irradiation (5.0 kGy) using scanning and transmission electron microscope to extend storage life of gills of Pleurotus ostreatus and Agaricus bisporus L. They found spore production was inhibited because of destruction of basidia rather than retardation of normal spore development. Beaulieu et al. (1999) treated Agaricus bisporus L. by 2 kGy ionization at different dose rate (4.5 kGy/h and 32 kGy/h). Both treatments showed a 2 and 4 days shelf life enhancement, respectively, compared to control.
2.7 Value addition
2.7.1 Mushroom preserve
Arumuganathan et al. (2005) prepared mushroom preserve. For this Button mushrooms were graded, washed, pricked and blanched in 0.05 per cent potassium metabisulphite for 5 minutes. It was treated with 40 per cent of its weight of sugar daily for three days. On the third day, mushrooms were taken out from the syrup and 0.1 per cent citric acid and remaining 40 per cent of the sugar was mixed in the syrup. After making its concentration to 65⁰Brix, mushrooms were added in the syrup. The prepared preserve was packed in plastic jar.
2.7.2 Mushroom candy
Arumuganathan et al. (2005) prepared mushroom candy. For this halved pieces of mushrooms were blanched for 5 minutes in 0.05 per cent of KMS solution. After draining for half an hour they were treated with sugar at the rate of 1.5 kg of sugar per kg of blanched mushroom. 0.1 per cent Citric acid is added to prevent the crystallization of sugar. Finally mushroom pieces were dried in a cabinet dryer at about 60⁰C for about 10
hours. As soon as these became crispy, all mushrooms were packed in polypropylene bags and stored in a cool and dry place for further analysis. Singh et al. (1994) prepared mushroom candy. It was prepared by placing blanched mushrooms in sugar syrup (2 parts sugar and 1 part water) with 0.2 per cent citric acid. Then draining out the sugar syrup after 20 days and drying the mushrooms in an oven at 30⁰C. Mean sensory score for candy was 30.
2.8 Preservation by sugar
According to Srivastava and Kumar (2002) syrups containing 66 per cent or more of sugar do not ferment. Sugar absorbs most of the available water with the result that there is very little water for the growth of microorganisms hence their multiplication is inhibited, and even those already present die out gradually. Dry sugar does not ferment. Thus sugar act as a preservative by osmosis and not as a true poison for microorganisms. Fruit syrup, jam, jelly, marmalade, preserve, candy, crystallized fruit and glazed fruit are preserved by sugar.




CHAPTER-3







Chapter 3 MATERIALS AND METHODS

This chapter deals with the description of raw materials, chemicals, instruments, experimental design and methodology used in the present investigation.
3.1 Source of Materials
Fresh white button mushrooms (Agaricus bisporus L.) were procured from Mushroom Research and Training Centre, G.B. Pant University of Agriculture and Technology, Pantnagar. Freshly harvested mushrooms were stored at 5±1⁰C till required for further processing. They were utilized within 24 hours of harvest. Sugar and artificial flavor were purchased from local market of Pantnagar.
3.2 Chemicals and Reagents
The chemicals and reagents used in the experiments, for chemical and microbiological analyses were of analytical/food grade.
3.3 Preparation of Mushroom Preserve
The mushrooms were procured during the morning hours, immediately after harvesting. They were sorted for any visible signs of microbiological infection, discoloration and physical injury. Undesirable mushrooms were removed during selection.
3.3.1 Washing
Casing soil and other foreign materials (straw etc.) from the surface of mushrooms were removed by washing them under tap water with gentle force to avoid any physical injury to tissues. They were then dipped in 0.1 per cent potassium metabisulphite solution for 5 minutes to prevent discoloration.
3.3.2 Standardization of Process Parameters
3.3.2.1 Method of preparation
Two different methods, viz, layering and syruping were used for the preparation of preserve. In layering method, alternate layers of mushroom and one third of sugar were placed in a container and allowed to stand for 24 hours. During this period, the mushrooms give out the excess of water, and the sugar goes into the pieces, resulting in syrup of 37-38⁰ Brix. More sugar (one third) was added to increase the strength of syrup to about 60⁰ Brix. On the third day, the strength of syrup was raised to 70⁰ Brix by adding rest of the sugar. In syruping method, mushrooms were placed in syrup of low sugar content (30 to 35⁰ Brix), made by boiling sugar in water, for 24-48 hours and then the concentration was raised to 68⁰ Brix in the final product. Appropriate concentration of sugar was selected on the basis of sensory characteristics.
3.3.2.2 Pricking treatment
Mushroom preserve was prepared by both pricking and non pricking method. In pricking method, selected, washed sound fruit bodies of uniform size were pricked by clean stainless steel fork. Pricking treatment is supposed to facilitates penetration of sugar syrup inside the mushroom pieces. The treatment was selected on the basis of sensory characteristics.
3.3.2.3 Blanching time
Mushrooms were blanched using three times their weight of boiling water for varying periods of 3-12 minutes, cooled in chilled water for 2 minutes and drained over muslin cloth for 1 minute. Blanched samples were analyzed for peroxidase and catalase activities. Optimum blanching time was selected on the basis of sensory characteristics and the time that gives negative test for peroxidase and catalase activities. This treatment was given to inactivate the enzymes responsible for discolouration of the product and thus it maintains whiteness of the processed mushrooms.
3.3.2.4 Preparation of sugar syrup
The sugar syrup was prepared with different concentrations (65⁰B, 68⁰B, 72⁰B) and the amount which gave best results was selected as the correct quantity of sugar, to be
Fresh button mushroom
Sorting and Grading
Washing in 0.05 % KMS
Pricking
Blanching
Cooling
Addition if one third of sugar and kept for 24 hours
Addition of next one third of sugar and kept for 24 hours
Removal of mushroom pieces from syrup
Addition of remaining one third sugar
Addition of 0.5 % citric acid
Heating the syrup up to 70⁰Brix
Cooling of syrup
Addition of mushroom pieces
Packing in bottles
Storage
Figure 3.1 : Flow diagram for preparation of mushroom preserve

added to preserve. The amount of sugar was standardized on the basis of sensory characteristics.
3.3.2.5 Amount of citric acid
Citric acid was added during the preparation to prevent crystallization of sugar syrup. Addition of citric acid improves both appearance and taste of the product. Different concentrations (0.2, 0.5 and 0.8 per cent) of citric acid were used and the concentration which gave best result was selected.
3.4 Study of storage stability of mushroom preserve
The mushroom preserve prepared from optimized process parameters was packed in clean, sterilized glass jars and stored at two different temperatures, that as room temperature (27±2⁰C) and at 35⁰C. The storage stability of the mushroom preserve was studied in terms of physico-chemical (Texture, percent acidity, pH, total soluble solids), microbiological (Total plate count and Yeast and Mold count) and sensory characteristics (appearance, colour, texture, taste and overall acceptability).
3.5 Analytical methods
3.5.1 Physical characteristics
3.5.1.1 Size of mushroom buttons
The total height, stalk height and cap diameter of mushroom buttons were measured using a Vernier Caliper (Least count 0.01 cm). The stalk diameter was measured using a Screw Gauge (Least count 0.001 cm). Average values of 10 mushrooms were calculated.
3.5.1.2 Weight
Ten randomly selected mushrooms were weighed on an electronic balance and average weight was calculated.
3.5.1.3 Specific gravity
The specific gravity of the mushrooms was expressed as the ratio of the weight in air of randomly selected mushrooms at a specific temperature (20 °C) to that of an equal volume of water replaced by the mushrooms when dipped in known volume of water taken in a measuring cylinder.
Hardness
Texture profile analysis of ‘Preserve’ was performed on texture analyzer (Stable Micro System Model TA-Xt2i) using 75 mm flat probe. Force calibration was carried out by using 25 kg load cell. The following test parameters were employed.
Test mode: (Texture Profile analysis): To perform first point of fracture.
Pre test speed: 5 mm/s
Test speed: 2 mm/s
Post test speed: 5 mm/s
Rupture test difference: 1mm
Distance: 20mm
Force: 0.98 N
Time: 5 s
The curve (force vs. time) gives the entire force history of the stimulated masticated action. Analysis of force time curve gave hardness or fracturability of mushroom preserve as force required causing first crack. Five replicate measurements were taken for each sample and average of three has been reported.
3.5.2 Chemical analysis
3.5.2.1 Moisture content
An empty aluminum dish was placed in an oven maintained at 105 ± 2°C for an hour. The dish was covered, cooled in a desiccator and weighed rapidly until constant weight was obtained. Five gram of sample was weighed accurately in this dish and it was kept in an oven maintained at 100 ± 2°C for a period of 12 hours. Drying was considered complete when readings of two consecutive weighings measured at an interval of an hour did not vary by more than 5 mg. Moisture content was calculated by deducting the dried weight from the fresh sample weight and was expressed as percentage on the basis of flesh (AOAC, 1995).
% Moisture content =
3.5.2.2 Crude Protein
Kjeldahl method was used to estimate protein content as described by AOAC (1995). Two gram sample was digested with five gram of digestion mixture (Potassium sulphate, copper sulphate and selenium oxide in the ratio of 100:20:2), and 40 ml of concentrated sulphuric acid. The contents were then digested till a clear blue green liquid was obtained. The volume of the digested material was made up to 100 ml with distilled water. A 20 ml aliquot of digested sample was distilled for about 20 minutes with 40 per cent sodium hydroxide solution. The ammonia liberated was absorbed in 2 per cent boric acid containing a few drops of mixed indicator (2 ml of 0.1 per cent methyl red, 10 ml of 0.1 per cent bromocresol green in 95 per cent alcohol), and the distillate was titrated against 0.1 N standard hydrochloric acid until the end point of light pink colour was obtained. Blank titration was carried by taking distilled water in place of sample.
Nitrogen content in the sample was calculated using the following formula:
(S-B) × N of HCl × 14 × V × 100
% Nitrogen = ––––––––––––––––––––––––––––––––––––––
v × W × 1000
1 ml of 0.1 N HCl = 1.4 ml N2
Where:
S = sample titre V = volume made up of the digest
B = Blank titre v = Aliquot of the digest taken
N = Normality W = weight of the sample taken
Conversion factor of 6.25 was used to convert per cent nitrogen to per cent protein.
Protein (%) = 6.25 × per cent nitrogen.
3.5.2.3 Crude fat
Soxhlet method was used to estimate crude fat content as per AOAC (1995) procedure. Twenty gram grated flesh sample was dried overnight in an oven maintained at 60 ± 2°C. The dried material was transferred to a thimble and plugged with cotton. The thimble was then placed in a Soxhlet apparatus and extracted with petroleum ether (B.P 40-60°C) (AR Grade) for about 16 hours. The extract was collected in a previously weighed oil receiver flask (Erlenmeyer flask). The solvent was removed by heating the oil flask on a water bath. It was dried in an oven at 60 ± 2° C to remove the residual solvent, cooled in a desiccator and weighed. The fat content of mushroom was also measured similarly by taking 2 g sample. The fat content was calculated as follows:
Fat (%) =(W_(3 -) W_1)/W_2 ×100
Where:
W1 = weight of oil flask
W2 = weight of sample taken
W3= weight of oil flask + fat
3.5.2.4 Carbohydrates (by difference)
Carbohydrates were calculated by the difference method (Ranganna, 1995) as per the under mentioned formula:
Carbohydrates (%) = 100 ― [moisture (%) + crude protein (%) +crude fat (%) + ash (%)]
3.5.2.5 Ash content
Ten gram of sample was accurately taken in a weighed tared silica dish which had been previously heated at about 600° C for 4 hours and cooled. The dish was heated first on a hot plate till all the material was completely charred. Thereafter, ashing was done in a muffle furnace at 550 ± 15° C for about 5 hours. It was then cooled in a desiccator and weighed rapidly. The ashing was repeated till it was almost white or grayish white in colour. The total mineral content was expressed as percentage on the basis of edible portion (AOAC, 1984).
Weight of residue
Ash (%) = ––––––––––––––––––––– × 100
Weight of sample

3.5.2.6 Crude fiber
Crude fiber content of the samples was determined according to AOAC (1984) procedure. Two gram of defatted sample was boiled in 1.25 per cent sulphuric acid for exactly 30 minutes, filtered through Whatman No.54 filter paper and washed with hot distilled water. The residue was then boiled in 1.25 per cent sodium hydroxide solution for exactly 30 minutes, filtered through Whatman No. 54 filter paper and washed with hot distilled water. The filter paper with the residue was dried in oven at 105⁰C for 3-4 hour till constant weight was obtained. The difference in weight of filter paper with residue minus filter paper was reported as crude fiber content of the sample and it was expressed as per cent crude fiber using the following formula:

3.5.2.7 Minerals
Preparation of ash solution
Ten gram sample was taken in a silica dish and heated to volatilize as much of the organic matter as possible. The dish was then transferred to a muffle furnace at 450⁰C for 7-8 hours. It was then removed from the muffle furnace, cooled and 40-50
ml diluted hydrochloric acid (1:1) was added. This dish was covered with a watch glass and heated over a water bath for 30 minutes, another 10 ml of dilute hydrochloric acid was added and heating was continued for another 30 minutes. It was then filtered into a 100 ml volumetric flask by using Whatmann No. 44 filter paper. The residue was washed by dilute hydrochloric acid and the volume was made up to 100 ml by distilled water (Ranganna, 1995).
Phosphorus
Phosphorus content of the sample was estimated according to the procedure described by Ranganna (1986). Five ml of ash solution and five ml molybdate reagent (25 g of ammonium molybdate dissolved in 400 ml of distilled water added to 500 ml of 10 N sulphuric acid and final volume made up to one litre with distilled water) were taken in a volumetric flask (50 ml). To this 2 ml of aminonaptholsulphonic acid solution (0.5 g 1-amino-2-napthol-4-sulphonic acid, 30 g sodium bisulphate and 6 g sodium sulphite, dissolved in 250 ml of water, left over night and filtered) was added and the volume was made up to 50 ml using distilled water. This was allowed to stand for 10 minutes and colour was measured at 650 nm by setting the blank at 100% transmission.
Standard curve: For standard, 0.439 g potassium
dihydrogen phosphate was dissolved in distilled water, 10 ml of 10 N sulphuric acid was added and the volume was made up to one litre. Ten ml of standard potassium dihydrogen phosphate solution was diluted to 50 ml with water (1ml = 0.02 mg P). Aliquots of this solution were pipetted from 5 to 40 ml into 50 ml volumetric flasks. 5 ml of molybdate reagent and 2 ml of aminonaptholsulphonic acid reagent were added and mixed. The volume was made up to 50 ml and colour was measured as in sample. A graph was plotted with concentration against absorbance.
Phosphorus content was calculated as follows:
w × V × 100
Phosphorus (mg/100g) = –––––––––––––––
v × W
w = mg of P in aliquot of ash solution used for estimation
V = Total volume of ash solution
v = Volume (ml) of ash solution taken for estimation
W = Weight of sample used for ashing.
Mineral estimation by Atomic Absorption Spectrophotometer (AAS).
The minerals viz., calcium, iron, manganese and zinc were estimated by atomic absorption spectrophotometer using the wet ashing procedure for preparation of ash solution as described by Raghuramulu et al. (2003) to eliminate organic materials and to free all the minerals into the solution.
Preparation of ash solution
Three gram of sample was weighed in a 100ml Kjeldahl flask and 25 ml mixture of concentrated nitric acid, perchloric acid and sulphuric acid in the ratio of 3:2:1 was added and shaken well for ensuring that no dry food lumps remain. Clean glass beads (acid washed) were added to the flask and left aside overnight in a fume cupboard. Thereafter, the flask was heated for about 30 minutes at 60⁰C continuously until the initial vigorous reaction had subsided. The flask was further heated until most of the nitrous fumes are removed. Heating was continued until white fumes of perchloric acid were evolved. A blank was also run simultaneously with each set of samples. After cooling, the contents of the digestion flasks were transferred quantitatively by 3-4 washings with distilled water to a 15 ml graduated test tube. The volume was made up to 10 ml mark with distilled water and mix thoroughly. The tubes were centrifuged for 30 minutes at 2000 rpm. The centrifuged solution was further diluted to 100 ml in a volumetric flask, filtered and stored in a cool place prior to elemental analysis by atomic absorption spectrophotometer.

Reading by AAS
A calibration curve of absorption against concentration was plotted by aspirating the samples of known concentration. Readings of absorbance were taken only after the instrument was set to zero with the use of blank. Samples were diluted prior to taking absorbance readings as in most of the cases the absorbance values were too high and did not fit into the range of the standard solutions. The particulars of mineral estimation by AAS like wavelength, slit width, flame used is presented in Table 3.1.
Table 3.1 : Particulars of mineral estimation by atomic
absorption spectrophotometer

Minerals Wavelength
(nm) Slit width
(nm) Flame used
Calcium 422.7 0.5 Nitrous oxide-acetylene
Iron 248.3 0.2 Air-acetylene
Manganese 279.5 0.2 Air-acetylene
Zinc 213.9 1.0 Air-acetylene
(Source: AAS 4141, Electronics Company of India Ltd.)
Mineral estimation by flame photometry
The potassium and sodium were estimated using flame photometer (128 make Systronic, microprocessor based) by method quoted by Ranganna (1986).
Representative sample in suitable liquid form was sprayed into the flame of a flame photometer and the absorption or emission of the mineral to be analyzed was measured at a specific wavelength.
Potassium
Potassium in solution is atomized into an oxyhydrogen or oxyacetylene flame. The flame excites atoms of potassium causing them to emit radiations at specific wavelengths. The amount of radiation emitted is measured in a spectrophotometer. Under standard conditions, it is proportional to the concentration of potassium in solution.
Reagents
Potassium chloride stock solution: Potassium chloride (1.909g) of AR grade was dissolved in distilled water and made up to 1 litre (1 mg potassium per ml or 1000 ppm).
Standard solution
One fifty ml stock solution (containing 150 ppm of potassium) and 5ml hydrochloric acid were dissolved in distilled water and solution was made up to 1 litre in order to compensate for minute interference produced by other ions in the flame photometry of potassium. It is recommended that standard solution be augmented with approximately equivalent concentrations of these ions that occur in highest proportions in
the samples being analyzed. Aliquots of standard solution were diluted from 0 to 150 ml making each aliquot to a volume of 150 ml with 0.5 per cent HCl. It was atomized, setting the top standard at 100 per cent transmittance. The luminosity of the flame for each concentration was noted.
Procedure
An aliquot of ash solution was diluted so that it contains less than 150 ppm potassium. Sufficient amount of HCl was added so that the concentration of the acid is same as in the standard solution. The diluted extract was atomized in a calibrated flame photometer with the wavelength at 768 nm.
Sodium
Sodium in solution is atomized into an oxyhydrogen or oxyacetylene flame. The flame excites atoms of sodium causing them to emit radiations at specific wavelengths. The amount of radiation emitted is measured on a spectrophotometer. Under standard conditions, it is proportional to the concentration of sodium in solution.
Reagents
Sodium chloride stock solution: Sodium chloride (2.5418g) of AR grade was dissolved in glass distilled water and volume made up to 1 litre(1 mg sodium per ml or 1000 ppm).
Standard solution
Ten ml of stock standard solution (containing 10 ml of sodium) and 5 ml HCl were measured into a flask and solution was made up to 1 litre using distilled water (this solution contains 10 ppm of sodium).
Procedure
An aliquot of ash solution was diluted so that it contains less than 10 ppm of sodium. Sufficient amount of HCl was added so that the concentration of the acid is same as in the standard solution. The diluted extract was atomized in a calibrated flame photometer with the wavelength at 589 nm.
3.5.2.8 Ascorbic acid
Ascorbic acid was determined by titration method using 2, 6 dichlorophenol Indophenol as described by Ranganna (1986).
Preparation of sample
Ten gram of sample was blended with an aqueous solution of metaphosphoric acid (3%) and the volume was made up to 100 ml with metaphosphoric acid solution. The content was filtered through a Whatman filter paper No. 1. Ten ml of the aliquot was titrated against dye solution till the appearance of light pink color.
Standardization of dye
Dye was standardized with freshly prepared standard ascorbic acid solution (0.1 mg/ml) prepared in 3 % metaphosphoric acid solution.
0.5
Dye factor = –––––––––––
Titre value
The content of ascorbic acid was expressed as milligrams of ascorbic acid present in 100 g sample.
Titre × dye factor× volume made up
Ascorbic acid = –––––––––––––––––––––––––––––––––– × 100
(mg/100 g) Aliquot taken × weight of sample
3.5.2.9 Sugars
The content of reducing sugars and total sugars was estimated by Lane and Eynon method (1923) as described by Ranganna (1986).
Preparation and standardization of Fehling Reagent:
Fehling’s reagent was prepared fresh by adding Fehling’s solution A in Fehling’s solution B in equal amount with constant stirring and the content was filtered through Whatman filter paper No.2. Ten milliliter of Fehling’s reagent was titrated against standard dextrose solution of concentration 2.5mg/ml using methylene blue as an indicator solution.
Titre × 2.5
Factor for Fehling’s solution = –––––––––––
1000
Preparation of sample
To prepare sample, 5 g of sample was transferred to a 100 ml beaker and the content was neutralized by adding 0.1 N NaOH using phenolphthalein as an indicator solution. It was added with about 50 ml of distilled water and the content was heated to boil. Thereafter, it was transferred to 250 ml volumetric flask using several washing with distilled water. Two milliliter of 45 per cent neutral lead acetate was added, stirred and allowed to stand for 10 minutes. It was added by adding predetermined amount of 22 per cent solution of potassium oxalate and the volume was made up to the mark with distilled water. Thereafter it was filtered through Whatman filter paper No.1.
Reducing sugars
The prepared sample was titrated with freshly prepared and pre-standardized Fehling’s reagent using methylene blue as an indicator solution. The content of reducing sugars was calculated as follows.
% Reducing sugar =
Total sugars
Twenty five grams of prepared sample was taken in a 100ml volumetric flask and 5 ml of HCl (1+1) was added. It was kept for 24 hours at the ambient temperature for the hydrolysis of non-reducing sugars to the reducing ones. The content was neutralized with 1N NaOH and made up to 100 ml with distilled water. It was titrated against freshly prepared and standardized Fehling’s reagent as described above.
% Total sugar =
Non-reducing sugars
The content of non-reducing sugars was calculated by the formula as:
% Non reducing sugars = 0.95 × (% total sugars – % reducing sugars)
Results were expressed as per cent of reducing, non-reducing and total sugars on the basis of sample.
Non enzymatic browning
To 5 g sample, 100 ml ethyl alcohol (60 per cent) was added. Sample was kept overnight; the content was thoroughly mixed and filtered through Whatman No. 1 filter paper. Optical density of filtrate was measured at 440 nm in spectrophotometer using 60 per cent ethyl alcohol as blank. The results were reported as absorbance values (Ranganna, 1986).
3.5.2.11 Titrable Acidity
Sample dispersion (10 g) was titrated against standard 0.1 NaOH using phenolphthalein as an indicator to a pink end point. The acidity was expressed in terms of per cent citric acid (AOAC, 1984).
Titre value × 0.6705
Acidity (as % citric acid) = –––––––––––––––––––––
Weight of sample
3.5.2.12 pH
Ten gram of sample was taken and volume was made to 50 ml with distilled water. The pH of dispersed material was measured using a digital glass electrode pH meter (Electronic Corporation of India, Ltd.).
Total soluble solids (TSS)

The total soluble solids of sample were determined using a hand refractometer (Erma Make, Tokyo, Japan). The prism was cleaned, and then covered with water. The instrument was adjusted to zero reading for water. After drying the prism, a few drops of sample was squeezed. The reading was corrected to 20 °C and the value was expressed as per cent total soluble solids.
Microbiological characteristics
The microbiological analysis was carried out according to the procedure given in A P H A (1992). One gram of sample was transferred aseptically to 9 ml of sterilized distilled water in culture tubes as dilution blank and mixed properly. Serial dilutions were also prepared. One milliliter of appropriate dilution was transferred to each of two sterilized petri dishes aseptically. 10 to 15 ml of pre-sterilized and melted nutrient medium as per the type of count was poured in each of the petri dish. After the solidification of medium, the petri plates were kept inverted in an incubator maintained at a specified temperature. The colonies were counted after the specific incubation period. The number of colonies was multiplied by corresponding dilution factor. After taking average, the viable count was expressed as colony forming unit (log cfu) per gram of the preserve.
Standard plate count
Total plate count was assessed according to the method as described in A P H A (1992) and standard plate count agar was used. Plates were incubated at 37 ± 2 °C for 1 to 2 days.
Yeast and Mold count
The yeast and mold count was conducted according to SP: 18, (Part I), ISI (1980) method. Potato dextrose agar acidified to pH 3.5 with sterile 10 per cent tartaric acid was used and incubation was carried out at 22 ± 2°C for 5 days.

Sensory characteristics
The sensory quality of mushroom preserve was evaluated using 9 point Hedonic scale where 9 and 1 represented liked extremely and disliked extremely, respectively (Amerine et al., 1965) as given below. The semi trained taste panel of 10 judges, comprising of students and the staff members of the Department of Food Science and Technology evaluated the products for different quality attributes using sensory cards (Appendix I) for color, taste, texture, appearance and overall acceptability. The mushroom preserve was evaluated between 11:00 A.M to 12:00 noon or 3:00 to 4:00 P.M.
Organoleptic score Rating
9 Liked extremely
8 Liked very much
7 Liked moderately
6 Liked slightly
5 Neither liked nor disliked
4 Disliked slightly
3 Disliked moderately
2 Disliked very much
1 Disliked extremely


Statistical analysis
The data obtained from sensory evaluation were analyzed
using Analysis of varience (ANOVA) technique of Snedecor and Cochran (1968) using completely randomized design (CRD). All the analysis was carried out at 1 per cent and 5 per cent levels of significance.


















CHAPTER-4







Chapter 4 RESULTS AND DISCUSSION

This chapter deals with the results of the experiments carried out to explore the possibilities of making value added product from mushrooms. The results obtained are presented and discussed as under following appropriate headings:
Morphological and physico-chemical characteristics of fresh button mushroom.
Standardization of ingredients and treatments for the preparation of mushroom preserve.
Physico-chemical, microbiological and sensory characteristics of mushroom preserve.
Effect of storage time and temperature on quality characteristics of mushroom preserve.
4.1 Morphological characteristics
The fresh button mushrooms were compact with veil closed and were white in colour (Fig 4.1). The features of button mushroom used in the present investigation were in accordance with specifications given by Lal and Sharma (1995).
4.2 Physico-chemical characteristics
The physical characteristics of button mushroom such as size, weight, stalk height, stalk diameter, cap height, cap


Figure 4.1: Fresh Button Mushrooms





Figure 4.2: Blanched Mushrooms
diameter are presented in Table 4.1. The proximate composition, mineral composition and other chemical constituents, namely, ascorbic acid and sugar contents are presented in Table 4.2, 4.3 and 4.4 respectively.
4.2.1 Physical characteristics
Size of button mushroom ranged from 32.4 to 43.7 mm with an average value of 34.75 mm whereas, weight ranged from 10.64 to 17.39 grams with an average of 14.45 g. The stalk height and stalk diameter ranged from 16.4 to 18.1 mm with an average of 17.6 mm and 11.28 to 16.5 mm with an average of 15.6 mm, respectively. The values for cap height and cap diameter varied from 16.2 to 17.8 mm (average 16.9 mm) and 28.6 to 30.4 mm (average 29.2 mm), respectively. The specific gravity value showed a range of 0.8-0.89 with an average value of 0.83. The values of size of button mushrooms were in the range of 25 to 45 mm as reported by Vaidya et al. (2008), however, the values of weight were higher than the values (11.5 ± 1.8 g) as reported by Beelman and McArdle (1975), Burton et al., (1987), Riva et al., (1991) and Lopez-Briones et al., (1992). The values of other parameters were in accordance with the values given by Vaidya et al. (2008). The values of specific gravity were slightly higher. The difference in values may be due to differences in growing conditions and strains.

Table 4.1: Physical characteristics* of fresh mushroom (Agaricus bisporus L.)


Parameter
Range
Average value**

Size (mm) 32.4-43.7 34.75
Weight (g) 10.64-17.39 14.45
Stalk height (mm) 16.4-18.1 17.6
Stalk diameter (mm) 11.28-16.50 15.6
Cap height (mm) 16.2-17.8 16.9
Cap diameter (mm) 28.6-30.4 29.2
Specific gravity 0.80-0.89 0.83
*on fresh weight basis
**average of 5 values









4.2.2 Proximate composition of fresh mushrooms
4.2.2.1 Moisture
Moisture content of button mushroom ranged from 91.35 to 92.62 with an average value of 91.68 per cent. These values were higher than that reported by Bano and Rajarathnam (1986) who gave a value of 89.5 %. The value were however, within the range (90.0-94.2 % and 92.8%) as reported by Sethi et al. (1991) and Pruthi et al. (1984).
4.2.2.2 Protein
The average protein content in button mushroom was 3.24 per cent (range 3.05 to 3.6 per cent) on fresh weight basis (Table 4.2). The values were in the range (3.1 to 3.9 per cent) as reported by Sethi et al. (1991), Anderson and Fellers (1942), Tomar (1998) and choudhary (2000).
4.2.2.3 Fat
The fat content was in the range of 0.29 to 0.35 per cent with an average value of 0.32 per cent. The values were in conformity with those reported by Sethi et al. (1991); Anderson and Fellers (1942); Tomar (1998) and choudhary (2000) found the fat content in the range of 0.28 to 0.92.
4.2.2.4 Carbohydrates
The average total carbohydrate content (by difference) of mushroom in the present investigation was found to be 3.68 per
Table 4.2: Proximate composition of fresh mushroom and mushroom preserve


Parameter (%)
Fresh mushroom*
Mushroom preserve*
Moisture 91.68 35.67
Protein 3.24 2.26
Fat 0.32 0.29
Total ash 1.08 0.61
Crude fiber 1.12 0.84
Carbohydrates 3.68 61.17
*on fresh weight basis

cent on fresh weight basis. The values fall in the range of 3.63 to 4.50 per cent as given by Sethi et al. (1991), Tomar (1998), choudhary (2000) and Tyagi (2004).
4.2.2.5 Total ash
The total ash content of button mushroom varied from 0.94 to 1.08 per cent. The values were in accordance with the values reported by Tomar (1998) and Choudhary (2000) who reported it in the range of 0.80 to 1.9 per cent.
4.2.2.6 Fiber content
The fresh button mushroom contained 0.98 to 1.23 per cent crude fiber (average of 1.12 per cent). The values were in the range (0.34 to 1.175 per cent) as reported by Li and Chang (1982), Tomar (1998), Sethi et al. (1991) and Choudhary (2000).
4.2.2.7 Minerals
The values of calcium, phosphorus, iron, sodium, potassium, manganese and zinc in fresh button mushrooms were 7.15 mg, 124 mg, 1.57 mg, 23.22 mg, 463.97 mg, 0.14 mg and 0.66 mg per 100g, respectively. These values were in accordance with the values reported by different workers in Table 2.5.
Table 4.3: Mineral content of fresh mushroom and mushroom preserve


Mineral
(mg/100 g)
Fresh mushroom*
Mushroom preserve*

Calcium 7.15 8.54
Phosphorus 124 90.23
Iron 1.57 2.83
Sodium 23.22 18.72
Potassium 463.97 227.12
Manganese 0.14 0.43
Zinc 0.66 0.78
*on fresh weight basis

Table 4.4: Chemical composition of fresh mushroom and mushroom preserve


Parameter
Fresh mushroom*
Mushroom preserve*
Reducing sugars (%) 0.49 2.1
Non-reducing sugars (%) 0.24 13.39
Total sugars (%) 0.75 16.2
Ascorbic acid (mg/100 g) 7.3 4.88
*on fresh weight basis




Figure 4.3: Optimized mushroom preserve




Figure 4.4: Optimized preserves stored at 35⁰ C and at room temperature

4.2.2.8 Ascorbic acid
Ascorbic acid content of fresh button mushroom was found to be 7.3 mg/100 g. The value obtained was in agreement with the value (5.69-8.69 mg/100 g) reported by Bano and Rajarathnam (1982), Sethi et al. (1991), Tomar (1998) and Choudhary (2000). However, it was higher than the value (5 mg/100g) reported by Pruthi et al. (1984).
4.2.2.9 Sugars
Reducing, non reducing and total sugars in fresh button mushroom were found to be 0.498 g, 0.239 g and 0.75 g per 100 g, respectively. The values were in accordance with those reported by Singh et al. (1999) and Kumar and Barmanray (2007).
4.3 Optimization of process parameters for preparation of mushroom preserve
4.3.1 Blanching time
The results showing the standardization of blanching time are shown in Table 4.5. Blanching was done to inactivate the enzymes catalase and peroxidase. The results showed that blanching of button mushrooms in boiling water for 3 minutes inactivated both the enzymes as catalase and peroxidase tests were negative. After that the blanching time was optimized on the basis of sensory evaluation and results shown in Table 4.5

Table 4.5: Sensory scores 1 of mushroom preserve prepared using varying periods of blanching

Blanching time (minutes) Appearance * Colour * Texture * Taste * Overall acceptability *

0 6.8a 7.4 7.1b 7.0a 7.0a
6 7.0a 7.4 7.5b 6.8a 7.3a
9 8.0b 7.3 8.2c 8.0b 8.1b
12 6.8a 7.3 6.4a 6.7a 6.8a


F-value ** NS ** ** **
CD at 5 % level 0.46 - 0.55 0.56 0.53
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance
**significant difference at 1 % level
NS: Non significant

Table 4.6: Sensory scores 1 of mushroom preserve prepared using different treatments

Treatment Appearance * Colour * Texture * Taste * Overall acceptability *

Pricking + Syruping 7.4a 7.4 6.4a 7.4a 7.2a
Pricking + Layering 7.8a 7.4 8.1b 7.9a 7.8b
Nonpricking + Syruping 6.7b 7.3 5.9b 6.4b 6.3c
Nonpricking + Layering 6.5b 7.4 6.4a 6.5b 6.4c


F-value ** NS ** ** **
CD at 5 % level 0.59 - 0.40 0.52 0.55
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance
**significant difference at 1 % level.
NS: Non significant


indicates that blanching for 9 minutes resulted in a product with maximum overall acceptability of 8.1 on 9 point hedonic scale. Blanching period of 9 minutes was found most acceptable on the basis of sensory attributes, namely, appearance, colour, texture and taste.
4.3.2 Treatment (Pricking and non-pricking)
Results of mushroom preserve made with and without pricking treatment are presented in Table 4.6. The results of sensory evaluation showed that the product prepared by pricking with layering method was most acceptable in terms of appearance, texture, taste and overall acceptability. However, no significant (P≤0.05) difference was observed in case of colour with respect to treatments. The significant (P≤0.05) difference was observed in the preserve made with or without pricking. Therefore, pricking treatment was found to be acceptable for the preparation of preserve.
4.3.3 Method of preparation
The mushroom preserve was prepared using layering method and syruping method. The samples were then evaluated for sensory attributes and the results are presented in Table 4.7. Significant (P≤0.05) difference was observed in mushroom preserves prepared using two different methods. The sensory scores were better for preserve made with layering method in


Table 4.7: Sensory scores 1 of mushroom preserve prepared using different methods

Method Appearance * Colour * Texture * Taste * Overall acceptability *

Layering 8.0b 7.4 8.0b 8.1b 7.4b
Syruping 7.1a 7.3 6.7a 7.1a 6.5a

F-value ** NS ** ** **
CD at 5 % level 0.89 - 0.63 0.72 0.56
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance
**significant difference at 1 % level
NS: Non significant

4.8: Sensory scores 1 of mushroom preserve prepared using varying concentrations of sugar

Concentration of sugar (⁰ Brix) Appearance * Colour * Texture * Taste * Overall acceptability *

65 6.8a 7.3 6.7a 6.4a 6.8 a
68 7.6b 7.3 7.4b 7.3b 7.6b
70 6.7a 7.4 6.5a 6.6a 7.0a

F-value ** NS ** ** **
CD at 5 % level 0.71 - 0.59 0.56 0.50
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance
**significant difference at 1 % level
NS: Non significant



terms of most sensory attributes, namely appearance, texture, taste and overall acceptability. However, the difference in colour was not significant at level of 5 per cent.
4.3.4 Standardization of recipe for mushroom preserve
4.3.4.1 Concentration of syrup
Sugar was added to mushroom preserve as a preservative agent to improve the shelf life of the product. Addition of sugar also improved the taste and overall acceptability of the product. Various levels of total soluble solids ranging from 65, 68 and 70⁰ Brix were used for the preparation of mushroom preserve. The samples were then subjected to sensory evaluation and the results obtained are presented in Table 4.8. The sensory scores for appearance, texture, taste and overall acceptability were maximum for TSS of 68⁰ Brix as compared to the product made using TSS of 65 and 70⁰ Brix. Therefore, 68⁰ Brix was found to be the optimum level of TSS for the preparation of mushroom preserve.
4.3.4.2 Amount of citric acid
Citric acid was added to mushroom preserve to prevent the crystallization of sugar and also to improve the taste of the product. Three different levels of citric acid (0.2, 0.5 and 0.8 per cent) were used. The results of sensory evaluation are presented in Table 4.9. The sensory scores for overall acceptability were

Table 4.9: Sensory scores 1 of mushroom preserve prepared using varying concentration of citric acid

Concentration of citric acid (%) Appearance * Colour * Texture * Taste * Overall acceptability *

0.2 6.5a 7.1 7.4 6.5a 6.8a
0.5 7.0b 7.0 7.4 7.5b 7.5b
0.8 7.8c 7.1 7.3 6.6a 6.8a


F-value ** NS NS ** **
CD at 5 % level 0.31 - - 0.318 0.27
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance
**significant difference at 1 % level
NS: Non significant




maximum for a preserve that contained 0.5 per cent citric acid. However, the sensory scores for appearance increased significantly (P≤0.05) with an increase in concentration of citric acid. The taste was also better in case of preserve prepared with 0.5 per cent citric acid, whereas, the effect of citric acid concentration on colour and texture was non significant. Hence, optimum concentration of citric acid was found to be 0.5 per cent.
4.4 Proximate composition of mushroom preserve
4.4.1 Moisture
Moisture content of mushroom preserve ranged from 34.98 to 36.20 with an average value of 35.67 per cent on fresh weight basis. Similar findings were reported by Sethi and Anand (1982). Who found moisture content of 35.5 per cent in amla preserve and 32.6 per cent in carrot preserve.
4.4.2 Protein
Protein content in mushroom preserve as presented in Table 4.2 ranged from 2.05 to 2.96 per cent with an average value of 2.26 per cent on fresh weight basis. Similar values (2.43 per cent) were observed in amla preserve by Sethi and Anand (1982).

4.4.3 Fat
Fat content in mushroom preserve as presented in Table 4.2 ranged from 0.25 to 0.30 with an average value of 0.29 per cent. As mushroom is a low fat food therefore lower values were obtained.
4.4.4 Carbohydrates
The average total carbohydrate content of mushroom preserve in the present investigation was found to be 61.17 per cent on fresh weight basis. The higher carbohydrates content in mushroom preserve as compared to fresh mushroom was due to added sucrose in the product.
4.4.5 Total ash
The average total ash content of mushroom preserve was found to be 0.61 per cent on fresh weight basis. Lower values (0.1 -0.29) were obtained by Sethi and Anand (1982) for carrot preserve. The variation might be due to differences in mineral composition of mushroom and carrot.
4.4.6 Fiber content
The mushroom preserve contained average fiber content of 0.84 per cent which are lower than that of fresh mushrooms. This may be due to solubilization of some fibers.
4.4.7 Minerals
The values of calcium, phosphorus, iron, sodium, potassium, manganese and zinc in mushroom preserve were found to be 8.54 mg, 90.23 mg, 2.83 mg, 18.72 mg, 227.124 mg, 0.43 mg and 0.78 mg per 100 g, respectively (Table 4.3). The values for calcium, iron, manganese and zinc were found to be slightly higher than in fresh mushrooms. This variation might be due to the mineral uptake from the utensil or due to the contamination with impurities present in the added water and sugar.
4.4.8 Ascorbic acid
Ascorbic acid content of mushroom preserve was 4.88 per cent on fresh weight basis (Table 4.4). A significant difference was observed in ascorbic acid values for fresh mushroom and mushroom preserve. The reduction in ascorbic acid content of mushroom preserve may be due to its oxidation into dehydroascorbic acid by oxygen (Fennema, 1976; Harris and Karma, 1977). Sethi and Anand (1982) also reported a significant reduction in ascorbic acid content of carrot and amla preserves.
4.4.9 Sugars
The values of reducing, non reducing and total sugar in mushroom preserve were found to be 2.1 g, 13.395 g and 16.2 g per 100g, respectively on fresh weight basis. The values were much higher than that observed for fresh mushrooms. The increase in values is due to the addition of sucrose in the preserve.
4.5 Effect of storage and temperature on mushroom preserve
After standardization of process parameters and recipe, the mushroom preserve was prepared from fresh mushrooms (Agaricus bisporus.L). The samples were stored in pre-sterilized glass jars at room temperature and at 35⁰C for 60 days. The effect of storage time and storage temperature on textural, chemical, microbiological and sensory characteristics are shown in Tables 4.10 to 4.17.
4.5.1 Textural characteristics
The results showing the effect of storage period and temperature on mushroom preserve are presented in Tables 4.10 and 4.11. The results showed a significant decrease in hardness up to 60 days of storage. The value of 140.76 Newton was observed on 0th day and thereafter, it showed a significantly high decline and reached to a value of 51.84 Newton. The decline in hardness was at a higher rate during initial stages (up to 30 days), thereafter, the decline was at a comparatively slower rate this may be attributed to the exosmosis which resulted in a loss of moisture. The adhesiveness reduced significantly from day 0
Table 4.10: Effect of storage time (days) on texture of mushroom preserve stored at room temperature


Parameter

Number of days


0 10 20 30 40 50 60

Hardness 140.76a 119.04b 104.67c 89.73d 77.09e 64.56f 51.84g
(Newton)

Adhesiveness -1.63a -1.91b -2.04c -2.08c -2.09c -2.16d -2.25e
Springiness 0.63a 0.58a 0.54c 0.51d 0.49e 0.47f 0.43g
Cohesiveness 0.44a 0.27b 0.26b 0.25b 0.23b,c 0.21c 0.19c
Gumminess 58.57a 47.83b 35.82c 27.07d 19.28e 15.66f 14.04f
Chewiness 35.92a 28.42b 19.67c 8.88d 6.56d 6.32d,e 5.27e
Resilience 0.24a 0.205b 0.114c 0.094d 0.085e 0.075f 0.068f

F-value CD at 5 % level
Hardness ** 3.708
Adhesiveness ** 0.055
Springiness ** 0.017
Cohesiveness ** 0.029
Gumminess ** 2.57
Chewiness ** 2.49
Resilience ** 0.0093

**significant difference at 1 % level

Table 4.11: Effect of storage time (days) on Texture of mushroom Preserve stored at 35⁰C


Parameter

Number of days


0 10 20 30 40 50 60

Hardness 140.76a 118.25b 106.58c 92.07d 86.06e 74.13f 64.22g
(Newton)

Adhesiveness -1.63a -1.89b -2.02c -2.41d -2.58e 2.66f -2.84g
Springiness 0.63a 0.59b 0.57c 0.52d 0.51e 0.49f 0.45g
Cohesiveness 0.44a 0.27b 0.24c 0.21c 0.19d 0.18d 0.18d
Gumminess 58.57a 40.85b 20.98c 17.54d 16.72d 15.15d,e 13.30e
Chewiness 35.92a 27.04b 21.09c 18.28d 16.40e 15.18e 14.56e,f
Resilience 0.24a 0.19b 0.12c 0.09d 0.08e 0.07e 0.06e.f

F-value CD at 5 % level
Hardness ** 2.58
Adhesiveness ** 0.067
Springiness ** 0.011
Cohesiveness ** 0.027
Gumminess ** 1.98
Chewiness ** 1.47
Resilience ** 0.0093




to day 60. The rate of decline was at a lower rate as compared to hardness. Springiness, cohesiveness, gumminess, chewiness and resilience also showed a significant decrease in values from the day it was prepared up to 60th day. Similar kind of trend was observed in both the samples kept at room temperature and at 35⁰C. Hence, no significant effect of temperature on textural quality of preserve was observed.
4.5.2 Chemical characteristics
The results showing the effect of storage period and storage temperature were presented in Tables 4.12 and 4.13 .The initial and final per cent acidity of mushroom preserve was 0.187 and 0.27, respectively, during storage of 60 days. The increase in acidity was significant (P≤0.05) at 5 per cent level. The initial and final pH after 60 days of storage was 3.91 and 3.35, respectively. The variation in pH during storage was significant (P≤0.05) at 5 per cent level. The decline in pH and increase in acidity may be attributed to the penetration of organic acids from acidulants present in the medium into the product. The total soluble solids showed highly significant difference from 0th day to 10th day, thereafter, the concentration of preserve was increased by heating the syrup to attain the equilibrium. After that, the values showed no significant variation up to 40 days, thereafter, significant difference was

Table 4.12: Effect of storage time (days) on Acidity, pH, TSS of mushroom preserve stored at room temperature


Parameter
Number of days


0 10 20 30 40 50 60

% Acidity 0.18a 0.20b 0.21c 0.22d 0.23e 0.25f 0.27g
pH 3.91a 3.81b 3.73c 3.63d 3.56e 3.41f 3.36g
TSS (⁰ Brix) 70.16a 70.00a 69.50b 69.50b 69.00c 68.50d 68.00e
**significant difference at 1 % level.
F-value CD at 5 %
Titrable acidity ** 0.0067
pH ** 0.029
TSS ** 0.191

Table 4.13: Effect of storage time (days) on Acidity, pH, TSS of mushroom preserve stored at 35⁰C

Parameter
Number of days


0 10 20 30 40 50 60

% Acidity 0.18a 0.20b 0.21c 0.23d 0.25e 0.26f 0.27g
pH 3.91a 3.81b 3.73c 3.63d 3.55e 3.40f 3.35g
TSS (⁰ Brix) 70.00a 70.00a 69.50b 69.17c 69.00c 68.67d 68.17e
**significant difference at 1 % level.
F-value CD at 5 %
Titrable acidity ** 0.0076
pH ** 0.024
TSS ** 0.33

observed up to 60 days (P≤0.05) at 5 per cent level. The same pattern of results was observed in both the samples stored at room temperature and 35⁰C.
4.5.3 Microbiological changes
The results of total plate count and Yeast and Mold count in terms of log of colony forming units (CFU) per gram of mushroom preserve are presented in Tables 4.14 and 4.15.
Total plate count was found to be 3.79, 1.06 and 0.74 log of colony forming units per gram on 0th, 10th and 20th day of storage of mushroom preserve stored at 35⁰C. Thereafter, the sample did not showed any growth up to 60 days of storage. Whereas, the sample stored at room temperature showed total plate count values up to 30 days of storage. The initial and final total plate count value for sample stored at 35⁰C was 3.79 and 1.59, respectively, as log of colony forming units per gram of sample. The results showed a significant (P≤0.05) decrease in the total plate count during storage. The data revealed that the initial and final Yeast and Mold count value was 3.06 and 0.71 and 3.06 and 0.77 log of colony forming units per gram of sample stored at room temperature and at 35⁰C, respectively. The results showed a significant (P≤0.05) decrease in the Yeast and Mold count during storage. The sample stored at room temperature had showed Yeast and Mold count up to 30 days of



Table 4.14: Effect of storage time (days) on microbiological quality of mushroom preserve stored at room temperature

Parameter
Number of days


0 10 20 30 40 50 60

Total plate count 3.79a 3.37a 2.11b 1.59c - - -
(log cfu/g)

Yeast and mold 3.06a 1.83b 1.23c 0.71d - - -
Count (log cfu/g)

**significant difference at 1 % level.
F-value CD at 5 %
Total plate count ** 0.45

Yeast and mold Count ** 0.43

Table 4.15: Effect of storage time (days) on microbiological quality of mushroom preserve stored at 35⁰C

Parameter
Number of days


0 10 20 30 40 50 60

Total plate count 3.79a 1.06b 0.74c - - - -
(log cfu/g)

Yeast and mold 3.06a 0.99b 0.77b - - - -
Count (log cfu/g)

**significant difference at 1 % level.
F-value CD at 5 %
Total plate count ** 0.51

Yeast and mold ** 0.26
Count


storage, whereas, up to 20 days of storage growth was observed in sample stored at 35⁰C. Thereafter, no counts were observed in both the samples till 60 days of storage. This may be attributed to the reduction in available water for the growth of microorganisms hence their multiplication is inhibited, and even those already present die out gradually Srivastava and Kumar (2002).
4.5.4 Changes in sensory quality
The samples of mushroom preserve stored at room temperature and at 35⁰C were evaluated on 9 point hedonic scale for sensory attributes, namely, appearance, colour, texture, taste and overall acceptability. The data obtained are presented in Tables from 4.16 and 4.17.
4.5.4.1 Appearance
There was no significant change in the appearance of mushroom preserve stored at room temperature, however, a significant change was observed in appearance of preserve stored at 35⁰C as depicted in Table 4.17 and 4.18. No significant variation in the appearance of preserve stored at 35⁰C was observed up to 30 days of storage, thereafter, a significant (P≤0.05) reduction was observed up to 60 days of storage. At the end of storage period appearance of both the products remained acceptable.

Table 4.16: Effect of storage time (days) on Sensory scores 1 of mushroom preserve stored at room temperature
No. of days Appearance * Colour * Texture * Taste * Overall acceptability *

0 7.70 7.25a 6.22a 6.65a 6.35a
10 7.87 7.35a 6.85b 7.17b 6.67b
20 7.90 7.50b,a 7.35c 7.80c 7.12c
30 7.95 7.35a,b 8.15d 8.05d 7.55d
40 7.95 7.15a,b 8.15d 8.15d 7.95e
50 8.05 7.05a,b 8.10d 8.15d 7.95e
60 7.82 7.05a,b 8.15d 8.07d 7.95e


F-value NS ** ** ** **
CD at 5 % level 0.25 0.24 0.27 0.18 0.16
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance.
**significant difference at 1 % level
NS: Non significant

Table 4.17: Effect of storage time (days) on Sensory scores 1 of mushroom preserve stored at 35⁰C
No. of days Appearance * Colour * Texture * Taste * Overall acceptability *

0 7.70a 7.25a 6.22a 6.65a 6.35a
10 7.75a 7.37a 6.52b 7.20b 6.82b
20 7.70a 7.25a 6.90c 7.77c,d 7.27c
30 7.62a 7.10a,b 7.35d 7.97d 7.57d
40 7.27b 6.52c 7.90e 8.00d 7.20c
50 6.87c 6.05d 7.92e 8.00d 7.07e
60 6.30d 5.75e 7.92e 7.95d 6.90b,e


F-value ** ** ** ** **
CD at 5 % level 0.27 0.23 0.19 0.20 0.18
1 Expressed on 9 point scale
*Means followed by different letters in a column differ significantly at 5 % level of significance.
**significant difference at 1 % level.
NS: Non significant


4.5.4.2 Colour
The sensory scores obtained for colour of mushroom preserve during storage of 60 days are presented in Tables 4.16 and 4.17. It is depicted that the decline in colour was significant (P≤0.05) for samples stored at both room temperature and at 35⁰C during storage. However, the decline was much more in sample stored at 35⁰C than that of sample stored at room temperature with a sensory score of 5.75 and 7.05, respectively. The variation in the colour of the preserve stored at room temperature and at 35⁰C might be due to non enzymatic browning which proceeds at a faster rate at higher temperature.
4.5.4.3 Texture
The variation in the texture was significant (P≤0.05) in mushroom preserve stored at room temperature and at 35⁰C as indicated in the Tables 4.16 and 4.17. The texture of sample stored at room temperature had showed a significant (P≤0.05) improvement up to 30 days of storage, thereafter, the variation was non significant. whereas, in case of sample stored at 35⁰C, the significant (P≤0.05) improvement was up to 40 days of storage and then the variation was non significant up to 60 days of storage.






4.5.4.4 Taste
The data presented in Tables 4.16 and 4.17 shows the variation in sensory scores for taste of mushroom preserve stored at room temperature and at 35⁰C. The sensory scores for both the samples showed a significant (P≤0.05) improvement in the taste of the product up to 60 days of storage. The improvement in taste may be due to the penetration of sugar syrup inside the pieces during the storage resulted in a uniform taste of the product.
4.5.4.5 Overall acceptability
The results shown in Tables 4.16 and 4.17 depicted that the changes in overall acceptability of mushroom preserve stored at room temperature and at 35⁰C were significant (P≤0.05) up to 60 days of storage. The sample stored at room temperature had showed gradual improvement up to 40 days followed by non significant difference up to 60 days of storage. Whereas, sample stored at 35⁰C, initially showed an improvement up to 40 days, thereafter a gradual fall was observed in the overall acceptability of the product up to 60 days of storage. But both the product remained acceptable after 60 days storage at room temperature.

4.6 Non enzymatic browning
The extent of non enzymatic browning was measured in terms of optical density for the mushroom preserves stored at room temperature and at 35⁰C. The higher values were observed in case of sample stored at 35⁰C than that at room temperature.














CHAPTER-5








Chapter 5 SUMMARY AND CONCLUSION

The present investigation was envisaged to standardize the process parameters for manufacturing preserve from fresh button mushroom (Agaricus bisporus L.). Fresh mushroom was studied for physico-chemical characteristics. The preserve prepared from button mushroom were evaluated for physico-chemical, microbiological and sensory characteristics. The products were kept for 60 days in pre- sterilized glass containers and were stored at room temperature and at 35⁰C. The samples were then analyzed for physico-chemical, microbiological and sensory characteristics at an interval of 10 days. The samples the specific findings are being summarized as follows:
Button mushrooms were almost white in colour. The size of mushroom had an average value of 34.75 mm with a range of 32.4 to 43.7 mm and weight ranged from 10.64 to 17.39 grams with an average of 14.45 g. The average value of stalk diameter, stalk height, cap height and cap diameter were 17.6 mm, 15.6 mm, 16.9 mm and 29.2 mm, respectively. The specific gravity ranged from 0.8 to 0.89 with an average value of 0.83.
The fresh mushroom had average value of 91.68 per cent moisture, 3.24 per cent protein, 0.32 per cent fat, 3.68 per cent carbohydrate, 1.08 per cent total ash and 1.12 per cent crude fiber. The calcium, phosphorus, iron, sodium, potassium, manganese and zinc were 7.15, 124, 1.57, 23.22, 463.97, 0.14 and 0.66 mg per cent, respectively. The fresh mushroom also contained 7.3 mg vitamin C, 0.498 g reducing sugars, 0.239 g non reducing sugars and 0.75 g total sugars per 100 g.
A blanching time of 9 minutes in boiling water was found optimum on the basis of enzyme (peroxidase and catalase) inactivation and sensory characteristics.
For optimum quality of mushroom preserve layering method, in addition to pricking treatment, with TSS of 68⁰ Brix and citric acid concentration of 0.5 per cent was found most suitable.
The mushroom preserve had average value of 35.67 per cent moisture, 2.26 per cent protein, 0.29 per cent fat, 61.17 per cent carbohydrate, 0.61 per cent total ash and 0.84 per cent crude fiber. The calcium, phosphorus, iron, sodium, potassium, manganese and zinc were 8.54, 90.23, 2.83, 18.72, 227.124, 0.43 and 0.78 mg per cent,
respectively. The mushroom preserve also contained 4.88 mg vitamin C, 2.1 g reducing sugars, 13.395 g non reducing sugars and 16.2 g total sugars per 100 g.
During storage of preserve, hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness and resilience, decreased significantly (P ≤ 0.05) up to 60 days of storage.
During storage of preserve, acidity, increased significantly (P ≤ 0.05) while the pH and total soluble solids, decreased significantly up to 60 days of storage in both the samples.
The initial total plate count of mushroom preserve was 3.79 log of colony forming units whereas Yeast and Mold count was 3.06 log of colony forming units. All these counts decreased significantly (P ≤ 0.05) with the advancement of storage (up to 60 days) in both the samples. However, in the later stages of storage, no counts were detected.
The sensory scores for overall acceptability, taste and texture of preserve showed a significant (P ≤ 0.05) increase during storage period at room temperature. However, overall acceptability of preserve stored at 35⁰C showed a decline. The sensory scores of appearance was non significant for sample stored at room temperature, whereas, sample stored at 35⁰C had shown a significant (P ≤ 0.05) decline with the advancement of storage. The sensory scores for colour also showed a significant (P ≤ 0.05) decline; however, the decline is more in sample stored at 35⁰ C than in stored at room temperature.
10.The extent of non enzymatic browning, measured in terms of O.D. was found higher in sample stored at 35⁰C than in sample stored at room temperature.
On the basis of above findings, it may be concluded that acceptable quality preserve could be prepared from fresh button mushroom after adding appropriate amount of sugar and citric acid. The product could be stored satisfactorily at room temperature and at 35⁰ C for a minimum of 60 days.









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APPENDIX - I

Department of Food Science and Technology
College of Agriculture
SENSORY EVALUATION CARD

Name : …………………………………………. Date : …………………..
Product: Mushroom Preserve Time : ………………….

Kindly evaluate the given samples for various quality attributes on the following scale:

Like extremely 9 Dislike slightly 4
Like very much 8 Dislike moderately 3
Like moderately 7 Dislike very much 2
Like slightly 6 Dislike extremely 1
Neither like nor dislike 5
Sample code Appearance Colour Texture Taste Overall acceptability


Suggestions/Remarks (if any)

(Signature)
APPENDIX - II
FPO Specifications for Preserves


Product kind and minimum minimum General characteristics
Variety of percentage percentage
Fruit of fruit of total soluble
portion in solids in the
the final final product
product (w/w)
(w/w)

Preserves Any 55 68 It may be a single or mixed preserve but
Fruit of fruit or vegetable used shall be mature,
Suitable fresh sound and clean. The only sub-
Variety stances that may be added are sugar,
dextrose, invert sugar or liquid glucose,
flavouring matter, citric acid, ascorbic
acid, permitted colours and preservatives.
The fruit shall retain form and shall be
permeated with the syrup without shrivel-
ing of the individual pieces. It shall be of
good keeping quality and attractive colour
and it shall be free from burnt and other
objectionable flavor, crystallization and
mold growth. The product shall not show
any fermentation, when examined. When
packed in cans, it shall show no positive
pressure at sea level.


When packed in sanitary top cans, the contents shall not be less than 85 per cent of total space of the can.







APPENDIX – III
Standard curve for phosphorus

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